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Farnesylation is a post-translational modification that promotes the interaction between the modified signaling protein and membrane lipids and/or other proteins. Farnesyltransferase is the crucial enzyme involved in this process. Strikingly, plant mutants in the ERA1 (Enhanced response to ABA 1) gene, encoding β-subunit of farnesyltransferase, exhibited ABA-hypersensitivity during seed germination and drought tolerance in several species including Arabidopsis, wheat and soybean. However, the mechanism of ERA1 action has not been resolved yet. Here, we present the potential regulatory role of ERA1 in the drought signaling network in barley. With the aim of decoding the role of the ERA1 gene, we developed a unique barley mutant using TILLING analysis. Mutation in HvERA1 confers semi-dwarf phenotype, ABA-sensitivity during seed germination and drought tolerance. Our transcriptomic analysis suggested a role of HvERA1 in regulation of the crosstalk between ABA and ethylene at the onset of drought. Furthermore, analysis of hvera1.b response to prolonged drought stress linked HvERA1 to the metabolism of galactolipids, that build the chloroplast membranes. It might results in the protection of hvera1.b photosystem and thus, in its better photosynthesis performance under water stress. Together, these results indicate the possible mechanism of the primary cause of the observed alterations in the hvera1.b mutant.

Physiological assays that facilitate screening for various types of responses to abiotic stresses are well established for model plants such as Arabidopsis; however, there is a need to optimize similar tests for cereal crops, including barley. We have developed a set of stress assays to characterize the response of different barley lines during two stages of development-seed germination and seedling growth. The assays presented, including the response to osmotic, salt, oxidative stresses, and exogenously applied abscisic acid, can be used for forward screening of populations after mutagenesis as well as for phenotyping of already isolated mutants, cultivars, or breeding lines. As well as protocols for stress treatments, we also provide methods for plant stress response evaluation, such as chlorophyll a fluorescence (ChlF) and image analysis.

Barley is one of the cereals that are most sensitive to aluminum (Al). Al in acid soils limits barley growth and development and, as a result, its productivity. The inhibition of root growth is a widely accepted indicator of Al stress. Al toxicity is affected by many factors including the culture medium, pH, Al concentration and the duration of the treatment. However, Al can act differently in different species and still Al toxicity in barley deserves study. Since the mechanism of Al toxicity is discussed we cytogenetically describe the effects of different doses of bioavailable Al on the barley nuclear genome-mitotic activity, cell cycle profile and DNA integrity. At the same time, we tested an established deep-water culture (DWC) hydroponics system and analyzed the effects of Al on the root system parameters using WinRHIZO software. We demonstrated the cytotoxic and genotoxic effect of Al in barley root cells. We showed that Al treatment significantly reduced the mitotic activity of the root tip cells and it also induced micronuclei and damaged nuclei. The DNA-damaging effect of Al was observed using the TUNEL test. We define the inhibitory influence of Al on DNA replication in barley. Analysis with the labelling and detection of 5-ethynyl-2'-deoxyuridin (EdU) showed that the treatment with Al significantly decreased the frequency of S phase cells. We also demonstrated that Al exposure led to changes in the cell cycle profile of barley root tips. The delay of cell divisions observed as increased frequency of cells in G2/M phase after Al treatment was reported using flow cytometry.

This work was supported by the European Regional Development Fund through the Innovative Economy for Poland 2007–2013, project WND-POIG.01.03.01-00-101/08 POLAPGEN-BD “Biotechnological tools for breeding cereals with increased resistance to drought,” task 22; National Science Centre, Poland, project SONATA 2015/19/D/NZ9/03573 “Translational genomics approach to identify the mechanisms of CBP20 signalosome in Arabidopsis and barley under drought stress.”, CBP20 (Cap-Binding Protein 20) encodes a small subunit of the cap-binding complex (CBC), which is involved in the conserved cell processes related to RNA metabolism in plants and, simultaneously, engaged in the signaling network of drought response, which is dependent on ABA. Here, we report the enhanced tolerance to drought stress of barley mutant in the HvCBP20 gene manifested at the morphological, physiological, and transcriptomic levels. Physiological analyses revealed differences between the hvcbp20.ab mutant and its WT in response to a water deficiency. The mutant exhibited a higher relative water content (RWC), a lower stomatal conductance and changed epidermal pattern compared to the WT after drought stress. Transcriptome analysis using the Agilent Barley Microarray integrated with observed phenotypic traits allowed to conclude that the hvcbp20.ab mutant exhibited better fitness to stress conditions by its much more efficient and earlier activation of stress-preventing mechanisms. The network hubs involved in the adjustment of hvcbp20.ab mutant to the drought conditions were proposed. These results enabled to make a significant progress in understanding the role of CBP20 in the drought stress response., European Regional Development Fund; National Science Centre, Poland

The bioinformatics resources provide a wide range of tools that can be applied in different areas of mutation screening. The enormous and constantly increasing amount of genomic data obtained in plant-oriented molecular studies requires the development of efficient techniques for its processing. There is a wide range of bioinformatics tools which can aid in the course of mutation discovery. The following chapter focuses mainly on the application of different tools and resources to facilitate a Targeting-Induced Local Lesions in Genomes (TILLING) analysis. TILLING is a technique of reverse genetics that applies a traditional mutagenesis to create DNA libraries of mutagenised individuals that are then subjected to high-throughput screening for the identification of mutations. The bioinformatics tools have shown to be useful in supporting the process of candidate gene selection for mutation screening. The availability of bioinformatics software and experimental data repositories provides a powerful tool which enables a process of multi-database mining. The existing raw experimental data (genomics-related information, expression data, annotated ontologies) can be interpreted in terms of a new biological context. This may help in selecting the proper candidate gene for mutation discovery that is controlling the target phenotype. The mutation screening using a TILLING strategy requires a former knowledge of the full genomic sequence of the gene which is of interest. Depending on whether a fully sequenced genome of a particular species is available, different bioinformatics tools can facilitate this process. Specific tools can be also useful for the identification of possible gene paralogs which may mask the effect of mutated gene. Bioinformatics resources can also support the selection of gene fragments most prone to acquire a deleterious nucleotide change. Finally, there are available tools enabling a proper design of oligonucleotide primers for the amplification of a gene fragment for the purpose of mutation screening.

Background Genetic studies on the molecular mechanisms of the regulation of root growth require the characterisation of a specific root phenotype to be linked with a certain genotype. Such studies using classical labour-intensive methods are severely hindered due to the technical limitations that are associated with the impeded observation of the root system of a plant during its growth. The aim of the research presented here was to develop a reliable, cost-effective method for the analysis of a plant root phenotype that would enable the precise characterisation of the root system architecture of cereals. Results The presented method describes a complete system for automatic supplementation and continuous sensing of culture solution supplied to plants that are grown in transparent tubes containing a solid substrate. The presented system comprises the comprehensive pipeline consisting of a modular-based and remotely-controlled plant growth system and customized imaging setup for root and shoot phenotyping. The system enables an easy extension of the experimental capacity in order to form a combined platform that is comprised of parallel modules, each holding up to 48 plants. The conducted experiments focused on the selection of the most suitable conditions for phenotyping studies in barley: an optimal size of the glass beads, diameters of the acrylic tubes, composition of a medium, and a rate of the medium flow. Conclusions The developed system enables an efficient, accurate and highly repeatable analysis of the morphological features of the root system of cereals. Because a simple and fully-automated control system is used, the experimental conditions can easily be normalised for different species of cereals. The scalability of the module-based system allows its capacity to be adjusted in order to meet the requirements of a particular experiment. Electronic supplementary material The online version of this article (doi:10.1186/s13007-016-0135-5) contains supplementary material, which is available to authorized users.